<Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> isolated from black tiger prawn larvae (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) in Vietnam

 A marine fungus was isolated from the black tiger prawn Penaeus monodon at Nha Trang, Vietnam, on March 20, 2001 and named isolate NJM 0131. The fungus was identified as Haliphthoros milfordensis from the characteristics of asexual reproduction, and its physiological characteristics were investigated. Although the optimum temperature for growth of the isolate was 25°–30°C, the fungus grew at a wide range of temperatures (15°–40°C). H. milfordensis grew well in 50%–100% seawater, but poorly in PYG agar containing 1.0%–5.0% NaCl and KCl. The fungus grew at a wide range of pH (4.0–11.0) with the optimum pH value of 7.0–9.0. The isolate also showed pathogenicity to swimming crab larvae (Portunus trituberculatus) by artificial infection, but mortality was not high. This is the first report of disease in the black tiger prawn P. monodon in Vietnam caused by H. milfordensis. Received: July 22, 2002 / Accepted: January 21, 2003 Correspondence to:K. Hatai     

Proteomic analysis of <Emphasis Type="Italic">Psychrobacter</Emphasis><Emphasis Type="Italic"> cryohalolentis</Emphasis> K5 during growth at subzero temperatures

It is crucial to examine the physiological processes of psychrophiles at temperatures below 4°C, particularly to facilitate extrapolation of laboratory results to in situ activity. Using two dimensional electrophoresis, we examined patterns of protein abundance during growth at 16, 4, and −4°C of the eurypsychrophile Psychrobacter cryohalolentis K5 and report the first identification of cold inducible proteins (CIPs) present during growth at subzero temperatures. Growth temperature substantially reprogrammed the proteome; the relative abundance of 303 of the 618 protein spots detected (∼31% of the proteins at each growth temperature) varied significantly with temperature. Five CIPs were detected specifically at −4°C; their identities (AtpF, EF-Ts, TolC, Pcryo_1988, and FecA) suggested specific stress on energy production, protein synthesis, and transport during growth at subzero temperatures. The need for continual relief of low-temperature stress on these cellular processes was confirmed via identification of 22 additional CIPs whose abundance increased during growth at −4°C (relative to higher temperatures). Our data suggested that iron may be limiting during growth at subzero temperatures and that a cold-adapted allele was employed at −4°C for transport of iron. In summary, these data suggest that low-temperature stresses continue to intensify as growth temperatures decrease to −4°C.     

Reaction of <Emphasis Type="Italic">Bipolaris euphorbiae</Emphasis> to environmental factors and its tolerance to solar and ultraviolet radiation

The determination of the ideal cultivation conditions and the knowledge of solar radiation tolerance are important aspects that must be investigated for the use of Bipolaris euphorbiae Muchovej and Carvalho as a biocontrol agent. The present study was done to assess the growth rate, sporulation and viability of the fungus cultivated under different conditions of the initial pH value of the growth medium, temperature and photoperiod. The tolerance of this fungus’ conidia to light from a source simulating solar and ultraviolet radiation was also assessed. B. euphorbiae was affected by different pH values of the growth medium being 6.0 the adequate pH for fungus cultivation. Incubation of the fungus under temperatures of 22, 25 and 28°C enhanced mycelium growth, while sporulation and viability of conidia were better at 22 and 16°C. Different regimens of exposure to light of fungal cultures only affect sporulation. Conidia exposed to solar and ultraviolet radiation for 8 h and 90 min, respectively, presented viabilities higher than 92%, indicating high tolerance levels to radiation exposure.     

Slow and steady phosphate solubilization by a psychrotolerant strain of <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">hepiali</Emphasis> (MTCC 9621)

A psychrotolerant phosphate solubilizing fungus has been isolated from the rock soil of a cold desert site in Indian Himalaya. The fungus grows from 4 to 35°C (optimum 21°C), and from 2 to 13.5 pH (optimum 9) under laboratory conditions. Based on phenotypic characters and 26S rDNA analysis, the fungus is identified as Paecilomyces hepiali. In quantitative estimation that was carried out at 9, 14, and 21°C, the fungus solubilized maximum phosphate at 14°C. In view of the slow growth and persistence of the desired activity at low temperature, the estimation was carried out for a longer period, i.e., up to 6 weeks. The suboptimal conditions for growth and biomass production were found to be optimal for phosphate solubilization by the fungus. At 14 and 9°C, the solubilization touched its maximum on day 42. Decline in pH was found to be significantly correlated with the phosphate solubilization at all the temperatures, under consideration. The acid phosphatase activity was found to be more prominent than alkaline phosphatase in culture filtrate. High performance thin layer chromatography (HPTLC) analysis showed production of six organic acids, gluconic and α-keto glutaric acid being in maximum amount in the culture filtrate. The study has ecological significance in view of the nutrient cycling under low temperature environment, prevalent in Himalayan region.     

Nutritional study on <Emphasis Type="Italic">Embellisia astragali</Emphasis>, a fungal pathogen of milk vetch (<Emphasis Type="Italic">Astragalus adsurgens</Emphasis>)

Embellisia astragali is a strong, virulent pathogen that develops within milk vetch (Astragalus adsurgens). In order to determine nutrient requirements, the fungus was cultured on 9 carbon sources, 9 nitrogen sources, and 13 growth media in the dark at 25°C. Growth rates and sporulation capacity were measured after 4 and 12 weeks. All carbon sources supported growth, but only soluble starch, inulin, and dextrose supported sporulation. In general, better growth was obtained on disaccharides and polysaccharides than on monosaccharides. Compared with no growth on NH₄ ⁺-N and urea, the fungus grew little on all NO₃ ⁻-N, amino-N, and other organic-N such as peptone. There was no sporulation or very sparse conidia on almost all nitrogen sources with supplied dextrose or soluble starch as sole carbon source. The better growth and sporulation on most of the semidefined media than on defined media indicates that some components in plant or animal material may be vital to the fungus. Sporulation was positively correlated with growth rate in N source experiment at 12 weeks and in growth media experiment at 4 and 12 weeks. The fungus favors grow within agar with growth rate less than 1.18 mm day⁻¹.     

Morphological,cultural, and biodestructive peculiarities of <Emphasis Type="Italic">Chaetomium</Emphasis> species

Chaetomium is a fungus species that inhabits different cellulose substrates. Some species of this genus destroy books, textile, and wood. This genus is of particular interest due to its antagonistic and enzyme peculiarities. Studies on morphological and cultural features of Chaetomium species have both theoretical and practical output. Growth rates and development of C. globosum, C. funicola, C. elatum, and C. spirale were studied under different temperatures (17–20°C, 25°C, 27°C, 30°C, and 33–35°C) and carbon source media (glucose, saccharose, mannite, lactose, amylum, and cellulose). The optimal growth temperature was 25–27°C for all the studied species, while the temperature range of 33–35°C inhibited the colonies’ growth. Growth dynamics and colony shape and morphology, as well as development of overhead mycelium and ascocarps, varied greatly in different Chaetomium species in regard to the carbon source media. When comparing the destructive effect of four studied species and C. murorum on craft paper, the highest activity was registered for C. globosum, C. funicola, and C. elatum.     

Plant growth-promoting activity in newly isolated <Emphasis Type="Italic">Bacillus thioparus</Emphasis> (NII-0902) from Western <Emphasis Type="Italic">ghat</Emphasis> forest,India

A Gram-positive rod-shaped bacterium isolated on nutrient agar plates incubated at 28 ± 2°C. The identity of the bacterium was confirmed by sequencing of the 16S rRNA gene and it reveals that it shares highest similarity with Bacillus thioparus CECT 7196^T (99.08%). It was capable of growing at temperatures ranging from 4 to 40°C, but optimum growth was observed at 28 ± 2°C. Strain NII-0902 is endowed with multiple plant growth promotion attributes such as phosphate solubilization, Indole acetic acid (IAA), siderophore and HCN production, which were expressed differentially at sub-optimal temperatures (5–40°C). It was able to solubilize phosphate (17.7 μg ml⁻¹), and produce IAA (139.7 μg ml⁻¹) at 28 ± 2°C. Qualitative detection of siderophore production and HCN were also observed. At 5°C it was found to express all the plant growth promotion attributes except HCN production. The ability to colonize roots is a sine qua non condition for a rhizobacteria to be considered a true plant growth-promoting rhizobacteria (PGPR). Bacillus sp. NII-0902 has a potential ability to colonize roots visualized by transparency, bacterial growth (turbid, milky and narrow zone) along and around roots and truly supported by scanning electron micrograph. Hence, it is proposed that, Bacillus thioparus sp. NII-0902 could be deployed as an inoculant to attain the desired results of bacterization.     

Effect of temperature on <Emphasis Type="SmallCaps">d</Emphasis>-arabitol production from lactose by <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

d-Arabitol production from lactose by Kluyveromyces lactis NBRC 1903 has been studied by following the time courses of concentrations of cell mass, lactose, d-arabitol, ethanol, and glycerol at different temperatures. It was found that temperature is a key factor in d-arabitol production. Within temperatures ranging from 25 to 39°C, the highest d-arabitol concentration of 99.2 mmol l⁻¹ was obtained from 555 mmol l⁻¹ of lactose after 120 h of batch cultivation at 37°C. The yield of d-arabitol production on cell mass growth increased drastically at temperatures higher than 35°C, and the yield reached 1.07 at 39°C. Increasing the cell mass concentration two-fold after 24 h of culture growth at 37°C, the d-arabitol concentration further increased to 168 mmol l⁻¹. According to the distribution of the metabolic products, metabolic changes related to growth phase were also discussed. The stationary-phase K. lactis cells in the batch culture that is started with exposing the precultured inoculum to high osmotic stress, high oxidative stress, and high heat stress are found to be preferable for d-arabitol production.     

A new species of <Emphasis Type="Italic">Discostroma</Emphasis> and its anamorph <Emphasis Type="Italic">Seimatosporium</Emphasis> with two morphological types of conidia,isolated from the stems of <Emphasis Type="Italic">Paeonia suffruticosa</Emphasis>

Conidia of two morphologically different types, one with a basal appendage only and the other with appendage at both ends, were isolated from the stems of Paeonia suffruticosa. Single conidial isolates of both types of conidia yield identical colonies, which then produced both types of conidia on agar media depending on temperature, thus showing that both types of conidia belong to the same fungus. Seimatosporium botan is described based on its morphological characteristics. The teleomorph of the fungus was first found on sterilized P. suffruticosa stems placed on water agar, when grown at 5°C for 2 months in 12-h photoperiod. Discostroma botan is described for this fungus. The teleomorph is also found on the same host in the field.     

Relationship between temperature and cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. <Emphasis Type="Italic">var. botrytis</Emphasis>) growth and development after curd initiation

Two experiments were conducted to assess the response of cauliflower (Brassica oleracea L. var. botrytis) cv. “Nautilus” F1 hybrid to different constant temperatures after curd initiation by keeping the plants in six different temperature-controlled glasshouse compartments with heating set point temperatures of 6, 10, 14, 18, 22, and 26 °C (±4 °C) at the School of Plant Sciences, The University of Reading, UK during winter 1998–1999 and summer 1999. Many of the growth parameters increased with increasing mean growing temperature up to an optimum temperature and then declined with further increases in temperature. Therefore, cauliflower’s growth and development after curd initiation could be resolved into linear or curvilinear function of effective temperatures calculated with optimum temperatures between 19 and 23 °C. It is suggested that future warmer climates will be beneficial for winter cauliflower production rather than summer cauliflower production.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

Resistance of the termite, <Emphasis Type="Italic">Coptotermes formosanus</Emphasis> Shiraki to <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> due to grooming

Termites, Coptotermes formosanus, reared individually, were highly susceptible to the entomopathogenic fungus, Metarhizium anisopliae, while termites reared in␣groups were highly resistant. When reared in groups, the termites treated with M.␣anisopliae conidia on the body surface were groomed by their nestmates and more than 80% of the conidia were removed from the cuticle within 3 h. However, there was not a significant reduction in the numbers of conidia on the body surfaces of termites reared individually. For the termites maintained in groups, conidia were found in foreguts, midguts and hindguts, but very few conidia were detected in the guts of termites reared individually. Conidia in the alimentary tracts did not germinate, but some of were alive. As a result, it seems that the removal of foreign bodies, such as fungal conidia, from the␣cuticle is one function of termite mutual grooming behavior and that conidia removed from the cuticle are eliminated through alimentary tracts. This study indicates that mutual grooming behavior is very effective in protecting these termites from M.␣anisopliae infection.     

Benzyladenine Promotes Flowering in <Emphasis Type="Italic">Doritaenopsis</Emphasis> and <Emphasis Type="Italic">Phalaenopsis</Emphasis> Orchids

Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m⁻²) containing BA at 100, 200, or 400 mg L⁻¹ or 25, 50, or 100 mg L⁻¹ each of BA and gibberellins A₄ + A₇ (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L⁻¹ had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L⁻¹ at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at 29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature.     

Optimum storage conditions for product of transiently expressed epitopes of <Emphasis Type="Italic">Human papillomavirus</Emphasis> using <Emphasis Type="Italic">Potato virus X</Emphasis>-based vector

We describe the optimized storage conditions of recombinant Potato virus A coat protein (ACP) carrying two different epitopes from Human papillomavirus type 16 (HPV-16). Epitope derived from minor capsid protein L2 was expressed as N-terminal fusion with ACP while an epitope derived from E7 oncoprotein was fused to its C-termini. The construct was cloned into Potato X potexvirus (PVX) based vector and transiently expressed in plants using Agrobacterium tumefaciens mediated inoculation. The effect of storage conditions on the serological activity of L2ACPE7 was studied by ELISA using IgG anti PVX, PVA and L2. Purified L2ACPE7 stored freeze-dried (at −20 °C), frozen at various temperatures (−20 °C, −70 °C) and at +4 °C were tested. Purified L2ACPE7 was most stable as lyophilized material stored at −20 °C. Our study demonstrates suitable way for the storage of plant material containing foreign viral epitopes for the purposes of edible vaccination.     

<Emphasis Type="Italic">In vitro</Emphasis> minimum growth for conservation of <Emphasis Type="Italic">Drosophyllum lusitanicum</Emphasis>

The present paper reports a protocol for minimum growth conservation of Drosophyllum lusitanicum (L.) Link. in vitro. Double-node cuttings were maintained for 4, 8 and 12 months at 5 or 25 °C in the dark. The effects of sucrose either alone at 5, 20, 30, 40 and 60 g dm⁻³ or at 20, 40 and 60 g dm⁻³ in combination with 20 g dm⁻³ mannitol, on survival and post-storage shoot multiplication efficiency were investigated. The cultures could effectively be conserved under minimum growth at 5 °C for 8 months on Murashige and Skoog’s medium supplemented with 60 g dm⁻³ sucrose, 20 g dm⁻³ mannitol and 0.91 μM zeatin. Following extended conservation, the cultures could be successfully regenerated into new shoots, and they were morphologically similar to those of non-stored controls.     

A comparative study of development and demographic parameters of <Emphasis Type="Italic">Tetranychus merganser</Emphasis> and <Emphasis Type="Italic">Tetranychus kanzawai</Emphasis> (Acari: Tetranychidae) at different temperatures

We investigated the effect of temperature on development and demographic parameters such as the intrinsic rate of natural increase (r _m) of the two spider mite species Tetranychus merganser Boudreaux and T. kanzawai Kishida at eleven constant temperatures ranging from 15 to 40°C at intervals of 2.5°C. Both male and female T. merganser and T. kanzawai completed development from egg to adult at temperatures ranging from 15 to 37.5°C. The longest developmental duration of immature stages was found at 15°C and the shortest developmental duration was found at 35°C for both species. Using linear and non-linear developmental rate models, the lower thermal thresholds for egg-to-adult (female and male) and egg-to-egg development were estimated as 12.2–12.3°C for T. merganser and as 10.8°C for T. kanzawai. The highest developmental rates were observed at around 35°C, whereas the upper developmental thresholds were around 40°C for both species. In fact, at 40°C, a few eggs of either species hatched, but no larvae reached the next stage. The r _m-values of T. merganser ranged from 0.072 (15°C) to 0.411 day⁻¹ (35°C), whereas those of T. kanzawai ranged from 0.104 (15°C) to 0.399 (30°C). The r _m-values were higher for T. kanzawai than for T. merganser at temperatures from 15 to 30°C, but not at 35°C (0.348 day⁻¹). Total fecundity of T. merganser was also higher than that of T. kanzawai at 35°C. These results indicate that higher temperatures favor T. merganser more than T. kanzawai.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Low-temperature limitation of primary photosynthetic processes in Antarctic lichens <Emphasis Type="Italic">Umbilicaria antarctica</Emphasis> and <Emphasis Type="Italic">Xanthoria elegans</Emphasis>

Temperature response curves of chlorophyll a fluorescence parameters were used to assess minimum sub-zero temperature assuring functioning of photosynthetic photochemical processes in photosystem II (PS II) of Antarctic lichens. Umbilicaria Antarctica and Xanthoria elegans were measured within the temperature range from −20 to +10°C by a fluorometric imaging system. For potential (F _V/F _M) and actual (Φ _II) quantum yields of photochemical processes the minimum temperature was found to be between −10 and −20°C. Non-photochemical quenching (NPQ) of absorbed excitation energy increased with temperature drop reaching maximum NPQ at −15°C. Image analysis revealed intrathalline heterogeneity of chlorophyll a fluorescence parameters with temperature drop. Temperature response of Φ _II exhibited an S-curve with pronounced intrathalline differences in X. elegans. The same relation was linear with only limited intrathalline difference in U. antarctica. The results showed that Antarctic lichen species were well adapted to sub-zero temperatures and capable of performing primary photosynthesis at −15°C.     

<Emphasis Type="Bold">Characterization of nitrile hydratation catalysed by </Emphasis><Emphasis Type="BoldItalic">Nocardia</Emphasis><Emphasis Type="Bold"> sp. 108</Emphasis>

Abstract Nocardia sp. 108 exhibited strong acrylonitrile-hydrating activity and its nitrile hydratase was Co²⁺-dependent. Nocardia sp. 108 was active within a broad pH range from 6.0 to 10.0 at 30°C and thermostable at temperatures below 35°C, but became unstable at temperatures above 45°C. Furthermore, it was found that Nocardia sp. 108 can hydrate indole-3-acetonitrile, p-chlorobenzonitrile, p-hydroxybenzylcyanide, 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, 3-cyanopyridine, o-chlorobenzonitrile to the corresponding amides and hence displayed a broad substrate specificity. The temperature and pH optima for these hydrations were 28°C and pH 7.0–7.5, respectively. At the observed concentrations, acrylonitrile was completely converted within 5 min, while 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, indole-3-acetonitrile, p-chlorobenzonitrile were approximately 21.71, 8.98, 34.44, 93.10% hydrated. p-Chlorobenzonitrile appeared to be the preferred aromatic nitrile for Nocardia sp. 108.     

The effect of temperature on growth characteristics and competitions of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Oscillatoria mougeotii</Emphasis> in a shallow,eutrophic lake simulator system

Blue-green algal blooms formed by Microcystis and Oscillatoria often occur in shallow eutrophic lakes, such as Lake Taihu (China) and Lake Kasumigaura (Japan). Growth characteristics and competitions between Microcystis aeruginosa and Oscillatoria mougeotii were investigated using lake simulator systems (microcosms) at various temperatures. Oscillatoria was the superior competitor, which suppressed Microcystis, when temperature was <20°C, whereas the opposite phenomenon occurred at 30°C. Oscillatoria had a long exponential phase (20 day) and a low growth rate of 0.22 day⁻¹ and 0.20 day⁻¹ at 15°C and 20°C, respectively, whereas Microcystis had a shorter exponential phase (2–3 days) at 30°C and a higher growth rate (0.86 day⁻¹). Interactions between the algae were stronger and more complex in the lake simulator system than flask systems. Algal growth in the lake simulator system was susceptible to light attenuation and pH change, and algae biomasses were lower than those in flasks. The outcome of competition between Microcystis and Oscillatoria at different temperatures agrees with field observations of algal communities in Lake Taihu, indicating that temperature is a significant factor affecting competition between Microcystis and Oscillatoria in shallow, eutrophic lakes.